Abstract
Publisher Summary The generation of a full-length complementary DNA (cDNA) can represent the most challenging part of a cloning project, particularly with respect to obtaining the 5' end of the cDNA. The initial step––reverse transcription of messenger RNA (mRNA)—is also the most critical one, and many variations on it is described to maximize the likelihood of obtaining fully extended cDNA. Thermal rapid amplification of cDNA ends (RACE) is a polymerase chain reaction (PCR) technique through which previously unobtained 3' and 5' ends of a cDNA can be amplified starting with the knowledge of a small stretch of sequence from within an internal region of the cDNA. This technique provides an alternative to constructing and screening conventional libraries to obtain the remainder of the sequence for a partially cloned cDNA. Partial cDNAs are generated using PCR to amplify the copies of the region between a single point in a mRNA transcript and its 3' or 5' end. To use the RACE protocol, a short, internal stretch of sequence must already be known from the mRNA of interest.
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